Karyotype and Chromosome Analysis

Karyotype

• A karyotype is the complete set of chromosomes in a specie.
• It describes the number of chromosomes and their appearance under a light microscope.
• It is a photographic representation of stained metaphase spread where chromosomes are arranged by decreasing length.
• Specimens with spontaneously proliferating cells include bone marrow, lymph nodes, solid tumors, and chorionic villi.
• Peripheral blood lymphocytes, tissue biopsies, and amniotic fluid samples are cultured to obtain dividing cells.
• Lymphocytes usually require the addition of a mitotic stimulant.
• Specimen selection for chromosome analysis depends on clinical indications and whether the diagnosis is prenatal or postnatal.

Chromosome Staining & Banding

• A chromosome band is a distinguishable part of a chromosome.
• It is comprised of alternating light and dark stripes.
• Banding and staining techniques can be categorized into two groups: those that produce specific alternating bands along the length of each chromosome and those that stain only a specific region.

C-Banding (Constitutive Heterochromatin Banding)

• Useful for determining the presence of dicentric and pseudodicentric chromosomes.
• Also useful for studying marker chromosomes and polymorphic variants.
• It selectively stains the constitutive heterochromatin around the centromeres.
• It stains inherited polymorphisms present on chromosomes 1, 9, and 16, and the distal long arm of the Y chromosome.

T-Banding (Telomere Banding)

• A harsher treatment of chromosomes diminishes staining except at the heat-resistant telomeres.

Polymerase Chain Reaction (PCR)

• Denaturation of dsDNA template occurs at 94℃.
• Two oligonucleotide primers anneal at 50-60℃.
• Polymerase extension of DNA molecules occurs at 72-74℃.

Standard 3-Step PCR Cycling

• Initial denaturation occurs at 94°C to 98°C for 1 minute.
• Denaturation occurs at 94°C for 10 to 60 seconds.
• Annealing occurs at 50°C to 60°C for seconds.
• Final extension occurs at 70°C to 80°C for 5 minutes.
• Cycles are typically repeated 25-35 times.
• Most thermal cyclers can pause at 4°C indefinitely at the end of the cycles.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

• Developed to amplify RNA targets.
• An RNA molecule is converted to a cDNA molecule via reverse transcriptase enzyme.
• The cDNA molecule is then utilized as a template sequence for the following PCR reaction.
• Its application is to detect RNA expression.

Nested PCR

• PCR is performed twice.
• The second round uses primers that bind to sequences within the product of the first reaction.
• Increases specificity and sensitivity by targeting a smaller and more specific region of the DNA.

Multiplex PCR

• Amplifies multiple DNA sequences in a single reaction.
• Uses different primer pairs for each target sequence.
• Allows for the detection of multiple genetic variations or pathogens simultaneously.

Digital PCR

• Quantifies the number of DNA molecules in a sample by partitioning it into thousands of individual reactions.
• Each reaction contains a single DNA molecule, which is then amplified and detected.
• Provides absolute quantification of DNA targets with high sensitivity and precision.

Quantitative PCR (qPCR) or Real-time PCR

• Monitors amplification in real-time.
• It monitors the amplification of a targeted DNA molecule during the PCR with use fluorescent dyes or fluorophore-containing DNA probes.

Electrophoresis

• A method of separating electrically charged substances in a mixture.
• A sample of the mixture is placed on a supporting medium to which an electrical field is applied.
• Each charged substance migrates toward the cathode or the anode at a speed that depends on its net charge, size, and shape.
• Biomolecules migrate towards electrodes of opposite charge due to electrostatic attraction.

Gel Electrophoresis

• A type of electrophoresis where the supporting medium is a gel.
• Separation occurs through molecular sieving based on the molecular size of the substances.
• The gel material acts as a "molecular sieve".
• Widely used for separating proteins and nucleic acids.

Two Methods of Gel Electrophoresis

• Horizontal Gel Electrophoresis
• Vertical Gel Electrophoresis

Types of Gels

• Agarose gel: Polysaccharide extracted from seaweed, casted horizontally, non-toxic, separates large molecules, commonly used for DNA separations, staining can be done before pouring the gel.
• Polyacrylamide gel: Cross-linked polymer of acrylamide, casted vertically, potent neurotoxic, separates small molecules, used for DNA or protein separation, staining can be done after pouring the gel.

Whole Genome Sequencing

• Determines the order of bases in the genome of an organism in one process.
• Uses DNA shearing, bar-coding, whole genome sequencing, and data analysis.
• DNA shearing cuts DNA into pieces small enough for the sequencing machine to read.
• Scientists add small pieces of DNA tags (bar codes) to identify which DNA piece belongs to which bacteria.
• The sequencer identifies the A’s, C’s, T’s, and G’s, or bases, that make up each bacterial sequence and uses bar codes to track which bases belong to which bacteria.
• Data analysis uses computer tools to compare bacterial sequences and identify differences.
• Allows for personalized disease treatment plans, cancer cell genotyping, and identifying previously unknown genes contributing to disease states.